engleski

Influence of growth conditions on the sphingoid bases biosynthesis in the yeast Candida lipolytica

Yeast's sphingolipids and their intermediates have diverse roles including signal transduction during the heat stress response, regulation of calcium homeostasis or components in calcium-mediated signalling pathways and regulation of the cell cycle. Since little is known about the regulation of synthesis of complex sphingolipids in any organism, investigating how sphingoid bases biosynthesis in the yeast can be regulated may give an answer to this question. The aim of our research is to establish how the growth conditions can influence the sphingoid bases biosynthesis. For this purpose, C. lipolytica was grown on synthetic medium containing glucose (1 %) as carbon source at 28 oC. To determine how growth phase can affect biosynthesis of sphingoid bases, cells were harvested in exponential (24 hours) and stationary phase (36 hours). The second approach to the research was to determine if the change in temperature would affect the biosynthesis. The cells were grown to the exponential phase, subjected to the shift in temperature from 28 oC to 39 oC for 40 minutes and then partially harvested. The remaining cells were grown further at 28 oC for the next 12 hours. Total sphingoid bases were extracted from the cells according to Riley et al. (Journal of AOAC International, Vol. 77, No. 2). O-Phthalaldehyde derivatives of the sphingoid bases were prepared and analysed by HPLC. The identity of the sphingoid bases was determined by comparing its elution profile with that of authentic standards (C18 phytosphingosine, C18 D-sphingosine, C18 DL- erythro- sphinganine and C20 sphinganine). According to the obtained results, the most abundant sphingoid base in C. lipolytica was C18 phytosphingosine, which accounted for approximately 24% of the total sphingoid bases. C20 sphinganine was present in the lowest amounts (less than 1%). Comparison of the concentrations of C18 phytosphingosine, C18 D-sphingosine and C18 DL- erythro- sphinganine determined in the exponential and the stationary phase cells showed that there was a 1.4-fold decrease in the concentrations of these sphingoid bases when cells entered the stationary phase of growth. Thus, more sphingoid bases in C. lipolytica were produced in the exponential than in the stationary growth phase. When cells were subjected to the heat shock immediately after the exponential phase, there was a 1.7-decrease in the concentrations of C18 phytosphingosine, C18 D-sphingosine and C18 DL- erythro- sphinganine in comparison with the exponential phase growth without heat shock. When such cells continued to grow further for the next 12 hours at 28 oC, there occurred a 1.5-fold increase in the concentrations of C18 phytosphingosine and C18 DL- erythro- sphinganine whereas the concentration of C18 D-sphingosine lowered. Comparison of these results with the ones obtained during the growth without heat shock showed that heat shock reduced sphingoid bases production in C. lipolytica and that their concentration was raised (except for C18 D-sphingosine) when the growth was returned to the normal conditions.

yeast; sphingolipids

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